Abstract Heparan sulfate proteoglycans (HSPGs), major components in the extracellular matrix (ECM) of all tissue types, participate in structural integrity of ECM and regulate cellular signaling via binding with ECM components and protein ligands such as growth factors and chemokines. Heparanase, the only known enzyme that can cleave the heparan sulfate (HS) side chains of HSPGs, regulates many cellular processes including ECM remodeling and homeostasis of cell- associated HS, and it controls the bioavailability and activity of molecules attached to HS. We hypothesize that heparanase cleaves HS side chains in a programmed manner ? certain HS cleavage leads to cell dissemination, while cleavage of other specific HS structures is in charge of release of specific signaling molecules, or homeostasis of HSPGs. Our long term goal is to define the precise role of heparanase in various pathological conditions. The current research focuses on the development of a set of molecular tools that can visualize spatiotemporal activities of heparanase in both live cells and animal models. These structurally defined probes incorporate in situ labeling strategy to retain the readout signals at site of heparanase action to achieve high spatial resolution and precision. We will use these molecular probes to test our hypothesis in cancer models, and to study the structurally defined role of heparanase during ECM remodeling using molecular imaging both in vitro and in vivo. !